This invention is generally in the area of delivery of agents for prevention of surgical adhesions, and specifically involves use of locally formed topical gel systems for controlled delivery of fibrinolysis enhancement agents, especially urokinase, for improved prevention of adhesions.
Adhesions in Surgery
The formation of adhesions, or scar tissue bridges, following surgery, remains a serious complication of many surgical procedures. These include pelvic, abdominal, spinal, tendon, ophthalmic, urinary, thoracic and other procedures. Adhesion formation is believed to occur through a series of events, one of which is the formation of fibrin bridges from a serosanguinous exudate occurring after surgery. The organs are first connected by thin fibrin bridges. Over time, these bridges become populated by cells, which may secrete collagen and otherwise stabilize the bridge. It has been observed that the level of cellular secretion of plasminogen activators, which normally cause the breakdown of fibrin by activating the enzyme plasminogen, can be decreased following injury to tissues. Thus, prevention of the stabilization of such fibrin bridges, and in particular enhancement of natural processes which can remove such bridges before their stabilization into adhesions, is highly desirable in the prevention of adhesions.
Cellular secretion of plasminogen activators, e.g., by the mesothelial cells that line the peritoneum, has been demonstrated by Vipond, et al., xe2x80x9cPeritoneal Fibrinolytic Activity and Intra-abdominal Adhesions.xe2x80x9d The Lancet, 335:1120-1122 (1990), to be reduced following injury, leading to the lack of resorption of the fibrin bridges prior to maturation into a scar. In the peritoneal cavity, the particular fibrinolytic reduced in secretion was demonstrated to be tPA and not uPA.
Barrier Methods in Adhesion Prevention:
Several physical barrier methods have been utilized in the prevention of postoperative adhesions. These include sheets of oxidized regenerated cellulose (U.S. Pat. No. 5,007,916 to Linsky and Cunningham; U.S. Pat. No. 5,134,229 to Saferstein, et al.) sheets of expanded polytetrafluoroethylene (Boyers, et al., xe2x80x9cReduction of Postoperative Pelvic Adhesions in the Rabbit with Gore-Tex(trademark) Surgical Membrane.xe2x80x9d Fertility and Sterility, 49:1066-1070 (1988)), thermoreversible hydrogels (U.S. Pat. No. 5,126,141 to Henry), and photopolymerized, resorbable hydrogels (U.S. Ser. No. 08/022,687 entitled xe2x80x9cPhotopolymerizable Biodegradable Hydrogels as Tissue Contacting Materials and Controlled-Release Carriersxe2x80x9d filed Mar. 1, 1993 by Hubbell, et al. The teachings of which are incorporated by reference herein). With the exception of the method of Hubbell, et al., these methods have ranged is usefulness, but in no case do the methods eliminate the formation of postoperative adhesions.
Use of Fibrinolytic enzymes in Prevention of Adhesions.
Various fibrinolysis enhancing agents have been used in attempts to prevent adhesions. Because of their availability and biological suitability, streptokinase (SK), urokinase (UK; also known as urokinase plasminogen activator, uPA), tissue plasminogen activator (tPA), and a modified recombinant tPA (Fb-Fb-CF) have been most widely tested. These agents all work by activation of the enzyme plasminogen, causing it to lyse fibrin. Other substances investigated for removal or prevention of fibrin strands have included proteolytic enzymes, drugs, and clotting inhibitors such as heparin, which tend to prevent deposition of additional fibrin, referred to herein as xe2x80x9cfibrinolysis enhancing agentsxe2x80x9d.
Fibrinolytic enzymes have been used in the prevention of postoperative adhesions, as reviewed by Dunn, xe2x80x9cTissue-type Plasminogen Activator and Adhesion Prevention.xe2x80x9d Prog. Clin. Biol. Res. 38:213-220 (1993), and xe2x80x9cAdhesion, Adhesiolysis and Plasminogen Activators.xe2x80x9d Assisted Human Reproductive Technology, 13:130-137 (1991).
Tissue Plasminogen Activator (tPA).
Tissue Plasminogen Activator (tPA) has been shown to be of use in the prevention of postoperative adhesions, when delivered by minipump infusion, intraperitoneal injection, and topically. Sheffield describes the topical administration of tPA preferably by injection, but possibly in a phospholipid carrier, a salve or ointment, a polysaccharide composition, a thermoplastic polymeric gel or a hydrogel such as a polyoxyethylene-polyoxypropylene block copolymer, which releases over a period of between three hours up to seven days. Wiseman, et al., describes the addition of tPA, a tPA analog, Fb-Fb-CF, and SK, alone or in combination with an absorbable sheet of oxidized regenerated cellulose, Interceed TC7 absorbable adhesion barrier from Ethicon, Inc., Somerville, N.J.
Streptokinase (SK).
Streptokinase (SK), the earliest plasminogen activator to become widely available, has been shown by some investigators to be effective in preventing adhesions and ineffective by others. SK has been shown to be effective in the prevention of postoperative adhesions when delivered by peritoneal injection (Meier, et al. xe2x80x9cFirst Clinical Results of Intraoperative Application of Streptokinase-Streptodornase in Children.xe2x80x9d Langenbecks Archiv. fur Chirurgie, 366:191-193 (1985), Treutner, et al. xe2x80x9cPostoperative, intraabdominelle Adhasionen-Ein neues standardisiertes und objektivierties Tiermodell und Testung von Substanzen zur Adhasionsprophylaxe.*xe2x80x9d Lagenbecks Archiv. fur Chirurgie, 374:99-104 (1989)) and ineffective in other studies when similarly delivered (Verreet, et al. xe2x80x9cPreventing Recurrent Postoperative Adhesions: An Experimental Study in Rats.xe2x80x9d Eur. Surg. Res., 21:267-273 (1989)). When delivered as a continuous infusion, it was not effective (Sheffield). When delivered from a degradable polymer matrix, it was somewhat effective (Wiseman, et al.).
Reports on side-effects of tPA differ widely, from none, at effective doses (Menzies and Ellis, or Sheffield), to severe at effective doses (Wiseman, et al.).
Fb-Fb-CF.
Fb-Fb-CF is a tPA analog (Phillips, et al., xe2x80x9cThe Effects of a New Tissue Plasminogen Activator Analogue, Fb-Fb-CF, on Cerebral Reperfusion in a Rabbit Embolic Stroke Model.xe2x80x9d Annals of Neurology, 23:281-285 (1989)) and was effective in the prevention of postoperative adhesions when released from a degradable polymer matrix (Wiseman, et al.).
Genetic engineering is being applied to generate additional forms of plasminogen activators, plasmin, plasminogen, and other fibrinolytic agents, for example, as described in U.S. Pat. No. 5,223,408, 5,185,259, and 5,094,953.
Urokinase Plasminogen Activator (uPA).
The majority of studies with Urokinase Plasminogen Activator (uPA) have not demonstrated usefulness in preventing surgical adhesions. uPA has been investigated in the prevention of adhesions, as reported by Dunn (1991). An initial study with uPA in several species was not successful. However, a second study by Gervin, et al., xe2x80x9cA Cause of Postoperative Adhesionsxe2x80x9d Am. J. Surg. 125:80-88 (1973), did show efficacy. Dogs were treated with an intraperitoneal injection of large amounts of urokinase at the time of surgery. At dosages of 20,000 U/kg, there was allegedly a significant decrease in the formation of ileal adhesions.
However, no subsequent studies have demonstrated efficacy. For example, as reported by Rivkind, et al. xe2x80x9cUrokinase Does Not Prevent Abdominal Adhesion Formation in Rats.xe2x80x9d Eur. Surg. Res., 17:254-258 (1985), who studied the administration of urokinase in dosages between 5,000 and 100,000 U/kg administered intravenously, intraperitoneally, and intragastrically immediately postoperatively and at 48-72 hours post surgery. Moreover, uPA released continuously with minipumps at the site of injury did not reduce adhesions, as reported by Sheffield, et al. Another study in rabbits claimed to show efficacy with abrasion injury to the uterine horns, following administration of 10,000 U/kg urokinase either intraperitoneally or intravenously at the time of surgery and at 24 and 48 hours after surgery, both when blood was added to the peritoneal cavity and without added blood (injury in both cases), Birkenfeld and Schenker xe2x80x9cThe Effect of Urokinase in the Prevention of Intraperitoneal Adhesions; Role of Blood in Their Formation.xe2x80x9d Annales Chirurgiae et Gynaecologiae, 72:246-249 (1983). Notwithstanding the assertion of efficacy, reference to Table 2 of Birkenfeld and Schenker, shows that comparison between the group xe2x80x9cSerosal abrading of the right uterine hornxe2x88x92No treatmentxe2x80x9d and the group xe2x80x9cSerosal abrading of the right uterine hornxe2x80x94Urokinase treatmentxe2x80x9d demonstrates a difference with a p value of only approximately 0.2. By contrast, the group xe2x80x9cSerosal abrading+application of blood on right uterine hornxe2x88x92No treatmentxe2x80x9d differs from the group xe2x80x9cSerosal abrading+application of blood on right uterine hornxe2x88x92Urokinase treatmentxe2x80x9d with a p value  less than 0.02, both by Chi-squared test. Thus, the treatment with urokinase was effective only with the addition of an unnatural amount of blood.
In summary, uPA has been shown to be effective in the prevention of adhesions in only one study, and it has not been possible to reproduce this observation using normal conditions and normal standards for statistical difference. Efficacy with other fibrinolytic agents has also been mixed, with the best results for tPA being obtained only with repeated or continuous infusion of high dosages, and essentially no efficacy being observed with streptokinase.
The literature in this field is highly equivocal about the utility of any of these agents as reliable, clinically effective means for prevention of surgical adhesions.
It is therefore an object of the present invention to provide a reliable means for locally preventing surgical adhesions using fibrinolytic agents.
It is a further object of the present invention to provide a means for preventing surgical adhesions which is minimally invasive, biodegradable over the same period of time as healing occurs, and simple to use.
It is still another object of the present invention to provide a means for preventing surgical adhesions which uses low dosages of fibrinolytic agents.
Described herein is a method of preventing adhesions by topical administration of fibrinolysis enhancing agents. In particular, described herein is a method using a local, degradable polymeric release system for the prevention of surgical adhesion using a topically applied polymeric matrix for delivery of a fibrinolytic agent, preferably urokinase, streptokinase, hirudin or ancrod. In the most preferred embodiment, the matrix is extremely thin and is polymerized in situ to form a biodegradable polymeric matrix. The matrix provides controlled release of the agent over a period of time effective to prevent surgical adhesions and is biodegradable, usually within the same time frame. In contrast to prior studies with urokinase administered systemically, the combination of the matrix and the urokinase is effective in preventing surgical adhesions, as shown by comparative examples. The examples also demonstrate efficacy for tissue plasminogen activator (tPA) administered via a controlled release, biodegradable system using low dosages.
Disclosed herein are delivery systems for the effective and reliable delivery of fibrinolysis-enhancing agents (FEAS) for the prevention of surgical adhesions.
Fibrinolysis-enhancing agents.
FEAs include agents such as urokinase, streptokinase, tissue plasminogen activator, hirudin, snake venom components such as ancrod, and genetically engineered variants thereof, all of which operate by binding to plasminogen and causing it to become proteolytically active against fibrin. FEAs also include thrombolytic agents; proteins directly attacking fibrin, such as proteases, including plasmin or plasminogen; molecules which inactivate a class of proteins which themselves inhibit plasminogen (anti-plasmin inhibitors); heparin and other fibrin-deposition blockers; and other agents having the effect of locally enhancing the dissolution of fibrin, or preventing its deposition.
An effective amount of one or more of these materials are administered topically to an area where adhesions are to be prevented. As used herein, xe2x80x9cadhesionsxe2x80x9d includes surgical or postsurgical adhesions, postinfection adhesions, strictures, scar tissue formation, and related processes. Adhesions can occur in pelvic, abdominal, spinal, tendon, ophthalmic, urinary, thoracic and other procedures.
In the preferred embodiment, the agent is urokinase or tPA, most preferably urokinase.
Dosages of FEAs are determined based on routine optimization from dosages currently in use as delivered systemically or by injection, or dosages that have been reported in the literature. In rats, tPA may be used in doses of 0.03 mg per animal up to doses of 12 mg per animal, in a treatment series; present evidence suggests a preferred range, when administered in a polymeric material, in the range of 1 to 2 mg per dose. Appropriate dosages for humans can be obtained by extrapolation; the above rat dose corresponds to the therapeutic amount normally administered for systemic fibrinolysis after heart attack, and thus may be near the upper end of the range. Urokinase likewise has proved effective at doses suitable for systemic application, about 45,000 International Units in the rat, and lower concentrations may be effective. The required range of streptokinase is likely to be higher than the equivalent of 15,000 IU per rat.
Means for topical administration.
Although a number of methods are known for topical delivery, those which have been discovered to be effective yield controlled release of incorporated FEAs over a period of time required to prevent adhesions from forming. The most effective and requiring least patient compliance are those consisting of a biocompatible biodegradable polymeric matrix which can be applied to the site where adhesions are to be prevented at the time of surgery.
The polymeric matrices should meet the following criteria:
The material should be biocompatible and biodegrade, either by hydrolysis or enzymatic cleavage, over a period of between a day and thirty days, for example, although longer degradation times may be desirable in some cases, as determined by the nature of the adhesions to be prevented and their location. In the abdomen, the preferred period is between one day and one month, preferably two days to two weeks, and most preferably, four days to one week.
The material should be in a form which is either (1) applied as a liquid or a gel, which is then polymerized or crosslinked in situ to provide additional physical integrity or binding to the tissue to be affected, or (2) be conformable and securable to the site where adhesion is to be prevented. These materials are distinguished from materials which are preformed before delivery to the site, such as Teflon(copyright) membranes, oxidized cellulose cloth, or gelatin sponges. The ungelled polymer may be delivered by any suitable means; the preferred means will vary with the particular situation in which adhesions are to be prevented, or fibrinolysis is to be enhanced. If the operation is conducted with laparoscope or trochar, then a preferred method is to spray an ungelled barrier material, preferably containing the fibrinolysis enhancing agent, onto the site of damage; and then to gel the barrier with light, or by other means suitable to the particular barrier system. If the operation involves an open wound, then it may be advantageous to apply the barrier by syringe or similar means. If the barrier is to be emplaced inside a hollow vessel or lumen, then use of a catheter for delivery may be most appropriate.
The thickness of the polymeric material will be determined by several factors, including the amount of FEA to be delivered, the time during which the FEA is to be delivered, the thickness of the barrier required for mechanical resistance, and similar factors. Within these limits, thinner barriers are preferred, since they will erode more rapidly, and often will be quicker to set, and are less likely to obstruct other organs or biological processes, such as blood flow. In general, the thickness contemplated are in the range of 1 to 500 microns. Ranges of 10 to 100 micron are preferred, and ranges of 20 to 50 microns are most preferred. However, thicker barriers are contemplated if required to contain or delivery the appropriate amount of FEA.
The erodability of the material is subject to control through selection of the composition and the stability of its degradable links. The requirements for stability of the material are that it persist long enough to achieve the desired effect, and that it then erode completely in as short a period as feasible. For the prevention of adhesions in the abdomen, current data suggest that the barrier, and the delivery of the FEA, should continue for at least one day, preferably for two or more, and most preferably for at least four days. In spinal disk surgery, fragmentary evidence suggests that periods of 1 to 2 weeks may be preferable, to prevent post-operative adhesions. It is preferable that the barrier complete its disintegration in less than a month, and more preferably in a shorter period such as two weeks, provided that this period is at least as long as the time required for the therapeutic presence of the FEA.
The material must also not be reactive with the incorporated FEA so as to inhibit their effectiveness, but must serve to control the rate of release of FEA at the site, preferably by diffusional restrictions. Since the time for healing is generally in the range of two weeks, the preferred time for release is in the range of between one day and up to thirty days. Control of diffusion is accomplished by design of pore sizes, relative affinity of the barrier matrix for the FEA, partial insolubility of the FEA, and physical size and shape of the deposited barrier. For short treatment times, of the order of a few days, the simple anticonvective effect of a porous hydrophilic matrix may give sufficient control.
The material may also function to prevent adhesion through properties of the polymer per se, which is cumulative to the action of the FEA. Such materials are described in U.S. Ser. No. 08/022,687 entitled xe2x80x9cPhotopolymerizable Biodegradable Hydrogels as Tissue Contacting Materials and Controlled-Release Carriersxe2x80x9d filed Mar. 1, 1993 by Jeffrey A. Hubbell, Chandrashekhar P. Pathak, Amarpreet S. Sawhney, Neil P. Desai, and Jennifer L. Hill, the teachings of which are incorporated herein. Briefly, polymers are formed from biocompatible, biodegradable, polymerizable and at least substantially water soluble macromers, having at least one water soluble region, at least one region which is biodegradable, usually by hydrolysis, and at least two free radical-polymerizable regions. The regions can, in some embodiments, be both water soluble and biodegradable. The macromers are polymerized by exposure of the polymerizable regions to free radicals generated, for example, by photosensitive chemicals and dyes.
An important aspect of the macromers are that the polymerizable regions are separated by at least one degradable region to facilitate uniform degradation in vivo. There are several variations of these polymers. For example, the polymerizable regions can be attached directly to degradable extensions or indirectly via water soluble nondegradable sections so long as the polymerizable regions are separated by a degradable section. For example, if the macromer contains a simple water soluble region coupled to a degradable region, one polymerizable region may be attached to the water soluble region and the other attached to the degradable extension or region. In another embodiment, the water soluble region forms the central core of the macromer and has at least two degradable regions attached to the core. At least two polymerizable regions are attached to the degradable regions so that, upon degradation, the polymerizable regions, particularly in the polymerized gel form, are separated. Conversely, if the central core of the macromer is formed by a degradable region, at least two water soluble regions can be attached to the core and polymerizable regions attached to each water soluble region. The net result will be the same after gel formation and exposure to in vivo degradation conditions. In still another embodiment, the macromer has a water soluble backbone region and a degradable region affixed to the macromer backbone. At least two polymerizable regions are attached to the degradable regions, so that they are separated upon degradation, resulting in gel product dissolution. In a further embodiment, the macromer backbone is formed of a nondegradable backbone having water soluble regions as branches or grafts attached to the degradable backbone. Two or more polymerizable regions are attached to the water soluble branches or grafts. In another variation, the backbone may be star shaped, which may include a water soluble region, a biodegradable region or a water soluble region which is also biodegradable. In this general embodiment, the star region contains either water soluble or biodegradable branches or grafts with polymerizable regions attached thereto. Again, the photopolymerizable regions must be separated at some point by a degradable region.
Examples of these macromers are PEG-oligolactyl-multiacrylates. The choice of appropriate end caps permits rapid photopolymerization and gelation; acrylates are selected because they can be polymerized using several initiating systems, e.g., an eosin dye, by brief exposure to ultraviolet or visible light. The poly(ethyleneglycol) or PEG central structural unit (core) is selected on the basis of its high hydrophilicity and water solubility, accompanied by excellent biocompatibility. A short poly a-hydroxy acid), such as polyglycolic acid, is selected as a preferred chain extender because it rapidly degrades by hydrolysis of the ester linkage into glycolic acid, a harmless metabolite. Although highly crystalline polyglycolic acid is insoluble in water and most common organic solvents, the entire macromer is water-soluble and can be rapidly gelled into a biodegradable network while in contact with aqueous tissue fluids. Such networks can be used to entrap and homogeneously disperse water-soluble drugs and enzymes, such as the FEAs, and to deliver them at a controlled rate. Other preferred chain extenders are polylactic acid, polycaprolactone, polyorthoesters, and polyanhydrides. Polypeptides and polysaccharides may also be used.
These materials are particularly useful for controlled drug delivery, especially of hydophilic materials such as most of the FEAS, since the water soluble regions of the polymer enable access of water to the materials entrapped within the polymer. Moreover, it is possible to polymerize the macromer containing the material to be entrapped without exposing the material to organic solvents. Release may occur by diffusion of the material from the polymer prior to degradation and/or by diffusion of the material from the polymer as it degrades, depending upon the characteristic pore sizes within the polymer, which is controlled by the molecular weight between crosslinks and the crosslink density. Deactivation of the entrapped material is reduced due to the immobilizing and protective effect of the gel and catastrophic burst effects associated with other controlled-release systems are avoided. When the entrapped material is an enzyme, the enzyme can be exposed to substrate while the enzyme is entrapped, provided the gel proportions are chosen to allow the substrate to permeate the gel. Degradation of the polymer facilitates eventual controlled release of free macromolecules in vivo by gradual hydrolysis of the terminal ester linkages.
An advantage of these macromers are that they can be polymerized rapidly in an aqueous surrounding. Precisely conforming, semi-permeable, biodegradable films or membranes can thus be formed on tissue in situ. In a particularly preferred embodiment, the macromers are applied to tissue having bound thereto a photoinitiator, and polymerized to form ultrathin coatings. This is especially useful in forming tissue barriers during surgery which thereby prevent adhesions from forming.
Examples in this application demonstrate the use of these macromers and polymers for the prevention of postoperative surgical adhesions in rat cecum and rabbit uterine horn models. The polymers show excellent biocompatibility, as demonstrated by minimal fibrous overgrowth on implanted samples. Hydrogels for the models were gelled in situ from water-soluble precursors by brief exposure to long wavelength ultraviolet (LWUV) light, resulting in formation of an interpenetrating network of the hydrogel with the mucous, serous, or serosanguinous layer coating the tissue. The degradable hydrogel was very effective, both by itself and in combination with tPA, in preventing adhesions.
These materials are distinguished from materials such as oxidized regenerated cellulose cloth, or perfluoroalkylene membranes, by their ability to be delivered by syringe, catheter, spray or solution, which enables their delivery to sites of tissue injury through less invasive techniques. Moreover, being biodegradable or bioerodable, they do not require removal. They are distinguished from liposomes, from polymeric adjuvants such as dextran, and from non-firmly gelled polymers such as hyaluronic acid, by their ability to be precisely localized at the site or sites where adhesion prevention is required. This minimizes the dose of FEA required to produce the desired effect.
Alternative polymeric materials include materials which are settable by other mechanisms. These include polymers which set on,warming, such as poloxamers (Pluronic(copyright) or Butyronic(copyright) detergents; block copolymers of polyethylene oxide and polypropylene oxide or polybutylene oxide), or hydroxypropyl methyl cellulose; polymers which gel on cooling, such as gelatin; polymers which can gel on exposure to physiological ions (calcium), such as alginate or chitosan; polymers which gel by other mechanisms, such as redox changes; provided in all cases that the settable polymers are also sufficiently inert and erodible.